The aims of this project are to assess the effects of aging and caloric restriction (CR) at a cellular and biochemical level of analysis, to identify physiological mechanisms associated with these effects, and to evaluate interventions/molecular pathways that might alter age-related declines in function. Laboratory studies consistently demonstrate extended lifespan in animals on calorie restriction (CR), where total caloric intake is reduced by 10-40% but adequate nutrition is otherwise maintained. CR has been further shown to delay the onset and severity of chronic diseases associated with aging such as cancer, and to extend the functional health span of important faculties like cognition. Less understood are the underlying mechanisms through which CR might act to induce such alterations. One theory postulates that CR's beneficial effects are intimately tied to the neuroendocrine response to low energy availability, of which the arcuate nucleus in the hypothalamus plays a pivotal role. CR induces measurable changes on circulating levels of several hormones and growth factors that regulate cell growth and proliferation. Serum obtained from CR animals alters growth, proliferation and stress responses of cells in culture. We have demonstrated that it is possible to investigate certain aspects of CR using this in vitro approach. This approach lends itself to a more rapid investigation of possible mechanisms and, perhaps more importantly to the research, development and rapid evaluation of interventions that would be able to induce or promote a phenotype similar to that seen with CR, essentially a CR mimetic. 1.- We are investigating the differential response to CR in male and female F1 offspring of DBA/2J and C57BL/6J mice. In a follow up to our published study 19, we wanted to determine if CR would have the same lifespan and healthspan benefits in the F1 offspring as it did with the parental strains of mice, and further investigate the mechanisms underlying these observations. In order to investigate the role of CR in extending lifespan in laboratory mice, the DBA2 strain, which is classically considered unresponsive to CR, and the standard C57BL6 mouse strain were subjected to 20% or 40% CR, and their response compared to ad libitum (AL)-fed mice 19. We found that male and female DBA2 mice were responsive to CR with maximum lifespan extension at 40% CR. In contrast, male and female C57BL6 mice responded very well to 20% CR, but there was no lifespan extension in 40% CR-fed females when compared to AL-fed C57BL6 mice 19. To test whether there was maternal inheritance, we crossed male and female offspring of DBA/2 dams x C57BL/6 fathers (D2B6F1) and C57BL/6 dams x DBA/2 fathers (B6D2F1) and maintained them on AL, 20% or 40% CR and tracked their survival (Figure 10). The study is still ongoing; however, preliminary results indicate that the B6D2F1 mice are unresponsive to CR, which is in contrast to previously published results 13. D2B6F1 mice respond best to 40% CR, with 20% CR having little to no effect in all F1 hybrids. 2.- We are also determining if the two different diets used in the NHP CR studies have the same outcomes in mice, in terms of longevity and health, and to investigate the underlying mechanism. In a novel approach to characterize the role of macronutrients in primates, we are comparing the short- and long-term effects of NIA and WIS NHP diets fed to mice in AL, pair-fed (PF) and CR regimens. This study is currently ongoing and expected to be finished within the next 3-6 months. In order to further understand the differences in survival and health outcomes and investigate the underlying mechanisms between the NIA and WNPRC diets, we established two pilot studies using mice and NHPs fed the NIA or WNPRC diets ad libitum. We found comparable metabolic effects in terms of bodyweight gain and fasting blood glucose over 3-4mo of diet between mice and monkeys (Figure 11), indicating the dietary effects are conserved across species. From these data, a longevity study was initiated with 4mo-old male C57BL/6J mice. Single-housed mice were started on either the WNPRC or NIA diet (Table 2), provided AL, 30% CR or restricted to a single isocaloric daily allotment (pairfed, PF) of food of the opposite diet at approximately AL levels in order to control for slight differences in caloric content between diets. CR and PF mice were fed a single daily allotment at approximately 2pm every day. Any food remaining the following day was removed, weighed and recorded to yield an accurate measure of daily energy consumption. Physiological, metabolic and behavioral measurements were collected across the study, including weekly body weights and temperatures, insulin and glucose homeostasis, metabolic cage, blood, and urine. A subset of mice was euthanized at 2 years of age and tissues collected for further analysis. A detailed histopathology assessment of the mice is underway by a board-certified veterinary pathologist. This study is ongoing, with a single NIA CR mouse still alive at 187 weeks of age. Preliminary survival curves are shown in Figure 12. The clear CR and PF effect indicates that reduction in calories (CR) and limited access to food (PF) extend mean and maximal lifespan in male mice relative to their AL controls. Cox proportional hazard regression was used to analyze differences in survival distributions for the type of diet (NIA vs WIS), feeding protocol (AL vs PF) and their interaction. Analyses were performed with PROC PHREG applications in SAS version 9.3. No analyses were performed for CR since mice is still alive. The results showed a significant main effect of feeding protocol (HR =1.65; 95% CI: 1.18 - 2.30; p = 0.0034) where AL-fed animals had a 65% higher hazard than mice on PF. The type of diet (p = 0.8981) and the diet-by-protocol interaction (0.9513) were not significant. Further analyses showed significant effects of the feeding protocol for animals on both diets, NIA (HR =1.63; 95% CI: 1.02 - 2.62; p = 0.0411) and WIS (HR =1.68; 95% CI: 1.04 - 2.73; p = 0.0354). These separate analyses illustrated that AL-fed animals had a 63% to 68% higher hazard ratios than the mice on the PF protocol, regardless of the diet, in agreement with the previous combined analysis. We assessed various measures of healthspan, including histopathology, and found important differences in the incidence and type of pathologies that mice died with. Notably, CR and PF protocols were able to delay the onset of pathologies in mice, whereas WIS-fed mice had a higher incidence of fatty liver compared to mice on NIA diet. This demonstrates that diet plays a role in the type of pathologies that an animal will develop, but the age of onset of the pathology and subsequent death are determined by the feeding regimen (CR, PF).